Conjugate consisting of an active substance and a non-exogeneous native protein

ABSTRACT

This invention relates to a conjugate which consists of an active substance and a native protein which is not regarded as exogenous and distinguishes itself in that an intracellularly cleavable linker is present between the active substance and the protein.  
     In addition, this invention concerns a process for the preparation of such a conjugate and its use.

[0001] This is a national phase filing of the Application No.PCT/DE95/01323, which was filed with the Patent Corporation Treaty onSep. 22, 1995, and is entitled to priority of the German PatentApplication P 44 33 890.2, filed Aug. 1, 1994.

FIELD OF THE INVENTION

[0002] This invention relates to a conjugate consisting of an activesubstance and a native protein which is not regarded as exogenous, aprocess for the preparation of such a conjugate as well as its use.

BACKGROUND OF THE INVENTION

[0003] For a long time there has been a great demand to transport inwell-calculated fashion pharmaceutical preparations to certain siteswithin the body where they are allowed to display their activity. Theformer has been achieved by an above conjugate. See, DE - 41 22 210. Itserves for concentrating a tumor-active compound in the tumor.

[0004] Surprisingly, it has now turned out that an above conjugate isalso highly active when an intracellularly cleavable linker is presentbetween the active substance and the native protein which is notregarded as exogenous.

[0005] Such a conjugate represents the subject matter of the presentinvention.

SUMMARY OF THE INVENTION

[0006] This invention relates to a conjugate which consists of an activesubstance and a native protein which is not regarded as exogenous anddistinguishes itself in that an intracellularly cleavable linker ispresent between the active substance and the protein.

[0007] In addition, this invention concerns a process for thepreparation of such a conjugate and its use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1 shows the binding of 4-aminophenylsulfonic acid or4-aminophenylphosphonic acid to albumin, an azo group being present aslinker.

[0009]FIG. 2 shows the binding of cytidine to albumin, a linkercontaining an azo group being present, and the release of aminocytidine.

[0010]FIG. 3 shows the binding of tetracycline to albumin, a linkercontaining an azo group being present.

[0011]FIG. 4 shows the growth inhibition of tumor cells byadministration of conjugates according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The above expression “active substance” comprises compounds ofany kind which can be used for treating a disease. They are, e.g.,compounds for treating tumoral, infectious and/or autoimmune diseases.Examples of such compounds are chemotherapeutic agents such asantibiotics, e.g., tetracyclines, and antimetabolites, e.g.,methotrexate, sulfonamides and nucleosides which after the incorporationinto a nucleic acid inhibit its replication and transcription,respectively. Preferred compounds of the above kind are those which havean acid group such as —CO₂H, —SO₃H, —PO₃H₂, or —AsO₃H₂. Especiallypreferred compounds are 4- and 2-aminobenzoic acid, 4- and2-aminophenylsulfonic acid, 4- and 2-aminophenylphosphonic acid, 4- and2-aminophenylarsonic acid as well as derivatives thereof. Furtherpreferred compounds are deoxyuridine (UDR), deoxycytidine (CDE),cytosine arabinoside (AraC), 5-fluorouracil (5-FU), 5-fluorodeoxyuridine(5-FUDR), azidothymidine (AZT).

[0013] Further examples of compounds as active substance are photoactivesubstances such as porphyrines, chlorines and bacteriochlorines whichcan be used for the photodynamic treatment.

[0014] One or several of the above compounds are present in a conjugateaccording to the invention. They are given as educts, which means thatthey are present in derivatized form in a conjugate according to theinvention. See, Examples 1-7 and FIGS. 1-3, infra.

[0015] An above active substance is linked via a linker to a protein.This protein is not considered exogenous by the body. It is alsoavailable in native, i.e., non-modified, form. In addition, the proteinhas a molecular weight (MW) of up to 90,000, preferably it is analbumin, particularly human serum albumin, or transferrin.

[0016] An above linker can be cleaved intracellularly. The expression“cell” comprises individual cells and cell aggregates. Examples of theformer are endogenous cells which are not present in an aggregate, e.g.,blood cells and virus-infected cells, and exogenous cells, e.g.,microorganisms such as bacteria, fungi and protozoa. Cell aggregatescomprise tissues, organs and tumors.

[0017] A person skilled in the art is familiar with a linker of theabove kind. He is also familiar with factors, e.g., enzymes, which causethe cleavage of certain chemical bonds in cells. Thus, he can constructfurther linkers which can be cleaved intracellularly. Such a linkerfavorably comprises an azo group, which is preferably cleaved. Thefollowing structure of the linker is especially favorable: wherein

—Y—R—N═N—

[0018] R is an organic group, preferably an aromatic one, and especiallypreferably phenylene or a derivative thereof, and

[0019] Y is a group selected from C(O), S(O)₂, P(O)OH and As(O)OH.

[0020] The above structure of a preferred linker corresponds to thatwhich the linker has in a conjugate according to the invention.Furthermore, at least when R is phenylene or a derivative thereof, thestructure comprises an active compound which is especially suited fortreating tumoral, infectious and autoimmune diseases. Having cleaved thelinker and optionally degraded the protein still linked to the linker,the compound can display its full activity. See, Examples 3 to 7 andFIGS. 2 and 3, infra.

[0021] Preferred conjugates of the present invention are shown in FIGS.1 to 3.

[0022] A process for the preparation of an above conjugate is alsoprovided according to the invention. In such a process, conventionalreactions occurring in chemistry such as diazotizing of an amino groupand activation of an acid group, are used individually or incombination. Reference is made to the preparation of the conjugates inExamples 1 to 7 and FIGS. 1 to 3.

[0023] Conjugates according to the invention distinguish themselves inthat they concentrate active substances in well-calculated fashion incertain cells of the body and allow them to fully display theiractivity. This is achieved by a combination consisting of a protein,e.g., albumin, and an intracellularly cleavable linker. Certain cells inthe body, particularly tumor cells, cells of flammable tissues andmicroorganisms, preferably absorb albumin and because of their enzymescleave the linker-active substance conjugate, so that the activesubstance and substances, respectively, is (are) released and can fullydisplay its (their) activity.

[0024] Thus, conjugates according to the invention are suited in thebest possible manner for therapeutic purposes, particularly for treatingtumoral, infectious and autoimmune diseases.

[0025] In addition, labelings (e.g., radioactive labelings) may exist inconjugates according to the invention, so that the conjugates can alsobe used for diagnostic purposes and therapy control, optionallysimultaneously for treatment.

[0026] The below examples explain the invention in more detail. Thefollowing preparations and examples are given to enable those skilled inthe art to more clearly understand and to practice the presentinvention. The present invention, however, is not limited in scope bythe exemplified embodiments, which are intended as illustrations ofsingle aspects of the invention only, and methods which are functionallyequivalent are within the scope of the invention. Indeed, variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intendedto fall within the scope of the appended claims.

EXAMPLES A. Example 1

[0027] Preparation of a Conjugate According to the Invention Consistingof Human Serum Albumin and 4-Aminophenylsulfonic Acid, an Azo GroupBeing Present as Linker

[0028] The preparation of the conjugate and its structure are shown inFIG. 1.

[0029] 1. Diazotization of 4-aminophenylsulfonic acid:

[0030] 4-Aminophenylsulfonic acid (173 mg, 1 mmole) was dissolved in 5ml 2 N HCl. The solution was cooled in an ice bath, and 600 μl of anice-cooled 2.5 M NaNO₂ solution (1.5 mmoles) were added in portions of0.1 ml each with constant stirring. After about 10 min, the excess ofnitrite was eliminated by the addition of urea.4-Diazoniumphenylsulfonic acid (4-DAPS) was obtained.

[0031] 2. Linkage of 4-DAPS to human serum albumin (HSA):

[0032] The 4-DAPS solution obtained under item 1. was slowly added to asolution of 2 g HSA in 30 ml of 0.17 M Bic in a molar ratio of 1:1 withpH control and constant stirring, so that the pH value was constantlyabove 7.5. During the addition of 4-DAPS already, the solution starteddyeing red, the color constantly increasing with proceeding reactiontime. Contaminations such as excess urea or salts were separated byultrafiltration via a YM 30 membrane in an Amicon pressure filtrationcell. A conjugate consisting of 4-aminophenylsulfonic acid and HSA wasobtained, an azo group being present as linker.

[0033] The purity of the conjugate according to the invention waschecked by means of HPLC (precolumn: Zorbax Diol 20μ (50×4 mm), column1: Zorbax GF 450, column 2: Zorbax GF 250, eluent: 0.2 M Na citrate, pH7.5, flow 1 ml/min).

B. Example 2

[0034] Preparation of a Conjugate According to the Invention Consistingof Human Serum Albumin and 4-Aminophenylphosphonic Acid, an Azo GroupBeing Present as Linker

[0035] The preparation of the conjugate and its structure are shown inFIG. 1.

[0036] The preparation was carried out as described in Example 1,4-aminophenylphosphonic acid having been used in place of4-aminophenylsulfonic acid.

C. Example 3

[0037] Preparation of a Conjugate According to the Invention WhichConsists of Cytidine, a Linker Containing an Azo Group and Human SerumAlbumin (Cytidine-4-DAPS-HSA)

[0038] The preparation of the conjugate and its structure are shown inFIG. 2.

[0039] 4-DAPS was prepared as described in Example 1.

[0040] 1. Linkage of 4-DAPS to cytidine:

[0041] 2.6 mmoles of cytidine (about 600 mg) were dissolved in 6 ml 2 NNaOH, and the 4-DAPS solution was added in portions (1 ml each) withstirring. The initially colorless cytidine solution adopts a more andmore intense red color during the addition of 4-DAPS already. Aftercompletion of the reaction, the deep-red solution was adjusted with 1 NHCl to a pH value of about 2 and then lyophilized. The dry residueobtained after the lyophilization is subsequently dissolved in a mixtureconsisting of 8 ml of methanol and 2 ml of DMF and separated frominsoluble sediment by filtration. 5(4-diazophenylsulfonicacid)cytidine(5(4-DAPS)cytidine) was obtained.

[0042] The purity of the product was checked by means of thin-layerchromatography (plates with fluorescence indicator, eluent: Etac/MeOH1/1).

[0043] 2. Activation of 5(4-DAPS)cytidine to form the corresponding HSIester:

[0044] An aliquot of the solution of 5(4-DAPS)cytidine was admixed inthe same solvent (4 parts of methanol and 1 part of DMF) with two timesthe molar amount of dicyclohexylcarbodiimide (DCC) and 7 to 10 times themolar amount of N-hydroxysuccinimide (HSI). After a reaction time ofabout 1 h, the activation of the 5(4-DAPS)cytidine into thecorresponding HSI ester is terminated. It can be used directly forlinkage to HSA.

[0045] 3. Linkage of the HSI ester of 5(4-DAPS)cytidine to HSA:

[0046] The HSI ester of 5(4-DAPS)cytidine was slowly added to a solutionof 2 g HSA in 30 ml 0.17 M Bic with constant stirring. DCC alreadyprecipitates during he addition of the HSI ester of 5(4-DAPS)cytidine.The turbid matter of DCC and DC urea was separated by means offiltration. Other contaminations such as methanol, DMF and HSI were thenseparated via a YM 30 membrane in an Amicon pressure filtration cell.Cytidine-4-DAPS-HSA was obtained.

[0047] The purity of the conjugate according to the invention waschecked by means of HPLC. See, Example 1, infra.

D. Example 4

[0048] Preparation of a Conjugate According to the Invention Consistingof UDR, a Linker Containing An Azo Group and Human Serum Albumin(UDR-4-DAPS-HSA)

[0049] The conjugate according to the invention was prepared asdescribed in Example 3, UDR having been used in place of cytidine.UDR-4-DAPS-HSA was obtained.

E. Example 5

[0050] Preparation of a Conjugate According to the Invention Consistingof AraC, A Linker Containing an Azo Group and Human Serum Albumin(AraC-4-DAPS-HSA)

[0051] The conjugate according to the invention was prepared asdescribed in Example 3, AraC having been used in place of cytidine.AraC-4-DAPS-HSA was obtained.

F. Example 6

[0052] Preparation of a Conjugate According to the Invention Consistingof CDR, a Linker Containing an Azo Group and Human Serum Albumin(CDR-4-DAPS-HSA)

[0053] The conjugate according to the invention was prepared asdescribed in Example 3, CDR having been used in place of cytidine.CDR-4-DAPS-HSA was obtained.

G. Example 7

[0054] Preparation of a Conjugate According to the Invention Consistingof 7-Chlorotetra-Cycline, a Linker Containing an Azo Group and HumanSerum Albumin

[0055] The preparation of the conjugate and its structure are shown inFIG. 3. 4-DAPS was prepared as described in Example 1.

[0056] 1. Linkage of 4-DAPS to 7-chlorotetracycline:

[0057] 718.5 mg (1.5 mM) of 7-chlorotetracycline (MW 478.9) weredissolved in 20 ml 1 N NaOH, and the 4-DAPS solution was added inportions (1 ml each) with constant stirring. The 7-chlorotetracyclinesolution initially dyed yellow adopted a more and more intense red colorduring the addition of 4-DAPS. After a reaction time of about 24 h, thedeep-red solution was adjusted to a pH value of about 2 using 1 N HCland lyophilized. The dry residue was then dissolved in a mixtureconsisting of 8 ml of MeOH and 2 ml of DMF and separated from theinsoluble sediment by filtration. 7-Chloro-9(4-diazophenylsulfonicacid)tetracycline(4-DAPS-chlorotetracycline) was obtained.

[0058] 2. Activation of 4-DAPS-chlorotetracycline for protein linkage:

[0059] An aliquot of the solution of the 4-DAPS-tetracycline was admixedin the same solvent (4 parts of MeOH and 1 part of DMF)—withoutpreceding separation of the excess 7-chlorotetracycline—with two timesthe molar amount of DCC (based on the employed amount of phenylsulfonicacid) and 7 to 10 times the molar amount of HSI. After a reaction timeof about 2 h, the activation of 4-DAPS-chlorotetracycline into thecorresponding HSI ester is terminated. The resulting ester can be useddirectly for protein linkage.

[0060] 3. Linkage of the HSI ester of 4-DAPS-chlorotetracycline to HSA:

[0061] The equimolar amount of HSI ester of 4-DAPS-chlorotetracycline isslowly added to a solution of 2 g of HSA in 30 ml 0.17 M Bic withconstant stirring. The excess of DCC precipitates already during theaddition of the HSI ester. The turbid matter of DCC and DC urea wasseparated by means of filtration prior to pressure filtration. Othercontaminations such as MeOH, DMF and HSI were separated via a YM 30membrane in an Amicon pressure filtration cell.7-Chloro-9(4-diazophenylsulfonic acid)tetracycline-HSA was obtained.

[0062] The purity of the resulting conjugate was determined by means ofHPLC. See, Example 1, infra.

H. Example 8

[0063] Preparation of a Conjugate Consisting of Tetracycline, a LinkerContaining an Azo Group and Human Serum Albumin

[0064] The conjugate was prepared as described in Example 7,tetracycline having been used in place of 7-chlorotetracyline. Thestructure of the conjugate is shown in FIG. 3.

I. Example 9

[0065] Growth Inhibition of Tumor Cells By Administration of ConjugatesAccording to the Invention

[0066] The conjugates UDR-4-DAPS-HSA (see, Example 4, supra),AraC-4-DAPS-HSA (see, Example 5, supra) and CDR-4-DAPS-HSA (see, Example6, supra) as well as HSA alone as control were incubated in each casewith Walker 256 cells under conventional conditions. The number of cellsper ml was determined after 24, 48 and 72 h, respectively.

[0067] As follows from FIG. 4, each of the conjugates according to theinvention reduces the proliferation of tumor cells as compared to thecontrol.

[0068] All references cited within the body of the instant specificationare hereby incorporated by reference in their entirety.

What is claimed is:
 1. A conjugate consisting of an active substance anda native protein which is not regarded as exogenous, characterized inthat an intracellularly cleavable linker is present between the activesubstance and the protein.
 2. The conjugate according to claim 1 ,characterized in that the active substance is a compound usable fortreating tumoral, infectious and/or autoimmune diseases.
 3. Theconjugate according to claim 2 , characterized in that the activesubstance is a chemotherapeutic agent and/or a photoactive compound. 4.The conjugate according to claim 3 , characterized in that thechemotherapeutic agent is an antibiotic.
 5. The conjugate according toclaim 3 , characterized in that the chemotherapeutic agent is anantimetabolite.
 6. The conjugate according to any one of claims 1 to 5 ,characterized in that several active substances are present.
 7. Theconjugate according to any one of claims 1 to 6 , characterized in thatthe linker comprises an azo group.
 8. The conjugate according to claim 7, characterized in that the linker has the following structure:—Y—R—N═N— wherein: R is an aromatic compound, and Y is a group selectedfrom C(O), S(O)₂, P(O)OH and As(O)H.
 9. The conjugate according to anyone of claims 1 to 8 , characterized in that the protein is albumin. 10.The conjugate according to claim 1 , namely the conjugate of FIG.
 1. 11.The conjugate according to claim 1 , namely the conjugate of FIG.
 2. 12.The conjugate according to claim 1 , namely the conjugate of FIG.
 3. 13.A process for the preparation of the conjugate according to claim 7 ,characterized in that the binding of the active substance via the linkerto the protein comprises a diazotization of amino groups and/or anactivation of acid groups.
 14. Use of the conjugate according to any oneof claims 1 to 12 for treating tumoral, infectious and/or auto-immunediseases.